Glutathione (GSH) is an essential component of antioxidant defense. Oxidative stress generated by quinones elevates synthesis and enzymatic activity of gamma-glutamylcysteine synthetase (GCS). This is due, in part to an increase in transcription of the catalytic (heavy) subunit of GCS. The mRNA for the regulatory (light) subunit of GCS is also increased. The increase in GCS enzymatic activity enhances the ability of cells to increase GSH synthesis, which may aid in producing greater resistance to oxidative stress from quinones as well as environmental toxins, such as nitrogen dioxide (NO2). The goal of this investigation is to determine the mechanisms of regulation and roles of the increased synthesis of GCS in oxidative stress. For these studies, we will use in vitro models of oxidant stress with epithelial cell lines derived from rat and human lungs. The specific aims are: (1) To identify the functional regulatory elements responsible for increased GCS in response to quinones by characterizing the promoter/enhancer region of the catalytic (heavy) subunit of GCS and dissecting the functional motif(s) modulating GSCS-HS expression, examining putative oxidative stress response elements of the promoter/enhancer of GCS-HS region using reporter constructs, and determining whether transcription and/or translation of the regulatory (light) subunit is coordinated with that of the catalytic subunit. (2) To determine the mechanism of oxidant signaling involved with the enhancement and sustained elevation of GCS by characterizing the oxidative stress responsive transcription factors involved with GCS-HS and examining the relationship of cellular H2O2 glutathione redox status (GSH/GSSG), glutathione-conjugates, and glutathione-protein mixed disulfides to synthesis of GCS heavy and light subunit mRNAs, proteins and enzymatic activity. (3) Determine the relationship between increased GSH synthesis and enhanced resistance to oxidative stress by examining whether oxidative stress from nitrogen dioxide and reactive aldehydes produce similar increases in GSH and GCS as observed with quinones and whether NO2, reactive aldehydes, and quinones can induce self tolerance or cross tolerance. The long range goal is the development of pharmacological approaches to elevating GSH that will provide protection against oxidative stress. This may extend to other pathologies, such as inflammation, where oxidative stress is a component.